Biofilms are three dimensional microbial growth forms comprising microbial communities and the extracellular matrix they produce. Biofilms are ubiquitous in nature, forming on any surface or at any interface where water or suitable fluid is available, or in suspension, for example as flocs or granules.
Biofilms are etiologic agents of a number of diseases and are associated with a variety of chronic infections in humans, forming on a variety of surfaces within the body, for example on surfaces in the respiratory tract and lungs (associated with cystic fibrosis and Legionnaire's disease), on surfaces of the ear (associated with otitis media), and on surfaces of the heart and heart valves (associated with bacterial endocarditis). Biofilms offer increased protection to the microorganism inhabitants, for example in the form of substantially increased resistance to antibiotics compared to planktonic cells and resistance to phagocytosis, which render biofilms very difficult to eradicate and explains the severity and high level of persistence of biofilms and the morbidity associated with infections produced by biofilms. In the case of cystic fibrosis, for example, a principal cause of respiratory infections is Pseudomonas aeruginosa, and P. aeruginosa biofilms on the surface of the lungs in cystic fibrosis sufferers imparts a greater degree of antibiotic resistance and resistance to host immune defences. Consequently the major cause of chronic lung infections, and in turn of morbidity and mortality, in cystic fibrosis sufferers is biofilm-associated P. aeruginosa. 
Biofilms also readily form on medical equipment such as catheters and cannulas, and on implantable medical devices including stents and contact lenses. Indeed many long term catheterization patients acquire infections caused by biofilm-forming bacteria, and more generally biofilms are responsible for a range of hospital acquired infections, adding considerable cost to health systems.
From a public health perspective, biofilms are important reservoirs of pathogens in water systems such as drinking water, reservoirs, pipes and air-conditioning ducts. Biofilms also cause significant industrial damage, causing, for example, fouling and corrosion in fluid processes such as water distribution and treatment systems, pulp and paper manufacturing systems, heat exchange systems and cooling towers, and contributing to the souring of oil in pipelines and reservoirs.
Biofilms are essentially multicellular microbial communities, the formation and development of which are dependent on various multicellular traits of the member organisms, such as cell-cell signalling. Extracellular signalling systems such as quorum sensing are used by bacteria to assess cell density and initiate changes in gene expression and phenotypes when sufficient concentrations of signalling molecules are reached. This is associated with differential gene expression, leading to the induction of, for example, virulence factors and/or defence mechanisms, and with cell differentiation such that biofilm-associated cells become highly differentiated from planktonic cells.
As the cells within biofilms differentiate and biofilms mature, reduced metabolic rates, the cellular expression of defence mechanisms and the reduced ability of antimicrobial agents to penetrate the biofilm results in increased antimicrobial resistance and make biofilms particularly difficult to eradicate. Present biofilm control strategies typically target the early stages of biofilm development and involve the use of toxic antimicrobial agents. However such toxic agents can present their own downstream problems, for example when used industrially due to their release into the environment. Improved strategies for biofilm control are clearly required.
Studies of P. aeruginosa, as well as other model biofilm forming bacteria, mixed species oral bioflims, and mixed species granular biofilms in waste water treatment processes have shown that programmed cell death induces detachment and dispersal of cells from biofilms (see, for example, Hope et al., 2002 and Webb et al, 2003) and is a general feature of biofilm development. Inventors of the present invention have previously found that programmed cell death in biofilms is linked to the accumulation of reactive oxygen and nitrogen species (RONS) within biofilm-forming organisms, and that programmed cell death and dispersal of cells from a biofilm into planktonic cells can be induced using low, non-toxic concentrations of nitric oxide generators or donors (see co-pending WO 2006/125262, the disclosure of which is incorporated herein by reference in its entirety).
The exploitation of this finding offers the prospect of novel technologies for the removal of biofilms in a broad range of environments and settings, including medical, industrial and bioprocessing by exposing biofilms to nitric oxide to induce the dispersal of cells. However in some settings, in particular in human health and medical applications, the uncontrolled and widespread release of nitric oxide may be associated with unacceptable side effects and toxicity levels. Improving the stability of nitric oxide donors in solution also poses a challenge. Accordingly, there is a need for the development of effective mechanisms to regulate, spatially and/or temporally, the release of nitric oxide such that this release can be localised in the vicinity of a biofilm to thereby minimise side effects and toxicity at other locations.
Now provided herein are compounds, methods and compositions for regulating the release of nitric oxide temporally and spatially and in turn providing novel mechanisms for promoting dispersal of cells from biofilms and regulating biofilm development.